Our overall goal is to understand the regulation of expression of multiple gap junction proteins (connexins) in vascular wall cells and their differing contributions to intercellular communication. There is an extensive family of connexins; in expression systems, different connexins form gap junctions with different channel properties and regulation. Therefore, we hypothesize that cellular coupling is regulated by the simultaneous or differential expression of multiple connexins in a single cell. Vascular endothelial cells and smooth muscle cells each express at least two connexins both in vivo and in vitro and provide an ideal system to test this hypothesis. First, we will use immunocytochemistry and in situ hybridization to determine the in vivo expression patterns of Connexin37, Connexin40, and Connexin43 in endothelium, smooth muscle and pericytes in different vascular beds. Tissue culture experiments will examine the biosynthesis and assembly of gap junctions formed of these proteins. We will compare the regulation of transcription, translation, and post-translational phosphorylation of these connexins. We will determine by immunocytochemistry and by biosynthetic studies and cross-linking whether these proteins can and do mix to form hybrid plaques or channels. Vascular gap junctions are modulated in response to changes in growth status. We will determine the specific effects of differing growth conditions and cytokines on transcription and translation of each of the connexins. Finally, we will determine the minimal promoter for Connexin43 and determine the genetic elements responsible for the growth or cytokine- related transcriptional regulation. Completion of these studies will provide new and valuable information on gap junction distribution and content in the vasculature, on assembly of gap junctions, on regulation of multiple connexins by cells, and on the regulation of connexin genes.